Isolation and Characterization of a Novel Gammaherpesvirus from a Microbat Cell Line

Shabman, Reed S.; Shrivastava, Susmita; Tsibane, Tshidi; Attie, Oliver; Jayaprakash, Anitha; Mire, Chad E.; Dilley, Kari E.; Puri, Vinita; Stockwell, Timothy B.; Geisbert, Thomas W.; Sachidanandam, Ravi; Basler, Christopher F.

Rapidly evolving next-generation sequencing (NGS) technologies can facilitate batvirus interaction studies both by identifying viruses that infect bats and by allowing a characterization of host responses to virus infection. For example, shotgun metagenomic studies have identified viral, bacterial, and eukaryotic sequences present in both environmental and animal samples (4, 5) . In this approach, sequencing is obtained directly from the sample source, and read data are then processed to identify sequence homology to known organisms through metagenomics analysis software tools (6) (7) (8) . A benefit of the shotgun metagenomics approach is that viruses unable to be propagated are readily identified. Alternative virus discovery approaches utilize degenerate primers specific for a virus family and/or random primers for signal amplification and subsequent NGS library construction. A recent study highlighting the power of the latter approaches used conserved primers across RNA and DNA virus families to sample the viral diversity in the Pteropus giganteus bat (9) . Sequencing of over 12,000 amplicons identified both known and novel virus sequences across nine virus families (9) . Transcriptomics and metatranscriptomics, analyzed historically through microarray and more recently by NGS, represent an approach that can both identify viral sequences in a given sample and characterize host responses to infections (reviewed in reference 10). Generally, mRNA is isolated from a sample, and postsequencing analysis allows for the identification of both host and viral mRNAs. This sequence-independent method allows for the identification of actively transcribed viral and host messages. Moreover, transcriptomic analyses by NGS from large DNA viruses can identify temporal gene expression patterns and transcriptional start and stop sites (11, 12) and can also define splice variants and novel protein coding transcripts (13) . A limitation of these approaches is that while they can rapidly identify genomic viral sequences, they often uncover only partial sequences (hundreds of bases of sequence or less) and rarely identify replication-competent virus. However, upon identification of viral sequences, approaches such as random priming and conserved primer approaches can be used to determine full-length viral genomic sequences, as illustrated by the discovery of severe acute respiratory syndrome (SARS) virus in passaged cultures obtained from infected individuals (14, 15) .